Dendritic Cell Maturation Transcription Factor E2F1 Suppresses

Transcription Factor E2F1 Suppresses Dendritic Cell Maturation Fang Fang, Yan Wang, Rui Li, Ying Zhao, Yang Guo, Ming Jiang, Jie Sun, Yang Ma, Zijia Ren, Zhigang Tian, Feng This information is current as Wei, De Yang and Weihua Xiao of September 29, 2021. J Immunol 2010; 184:6084-6091; Prepublished online 26 April 2010; doi: 10.4049/jimmunol.0902561

http://www.jimmunol.org/content/184/11/6084 Downloaded from

References This article cites 40 articles, 21 of which you can access for free at: http://www.jimmunol.org/content/184/11/6084.full#ref-list-1 http://www.jimmunol.org/

Why The JI? Submit online.

• Rapid Reviews! 30 days* from submission to initial decision

• No Triage! Every submission reviewed by practicing scientists

• Fast Publication! 4 weeks from acceptance to publication by guest on September 29, 2021

*average

Subscription Information about subscribing to The Journal of Immunology is online at: http://jimmunol.org/subscription Permissions Submit copyright permission requests at: http://www.aai.org/About/Publications/JI/copyright.html Email Alerts Receive free email-alerts when new articles cite this article. Sign up at: http://jimmunol.org/alerts

The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2010 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Transcription Factor E2F1 Suppresses Dendritic Cell Maturation

Fang Fang,*,1 Yan Wang,*,1 Rui Li,* Ying Zhao,* Yang Guo,* Ming Jiang,* Jie Sun,* Yang Ma,* Zijia Ren,* Zhigang Tian,* Feng Wei,† De Yang,†,‡ and Weihua Xiao*

Transcription factor E2F1 has been largely studied as a promoter of S-phase transition in the cell cycle and as a regulator of apoptosis. Recently, E2F1 has been shown to regulate a wide range of genes in response to inflammatory stimulation of macrophages and to contribute to T cell activation in response to pathogens, implicating an extensive immunological role for E2F1. Dendritic cells (DCs) play critical roles as professional APCs in the development of immune responses. However, it is unclear whether E2F1 has any effect on DC phenotype or function. In this paper, we report that E2F1 acts as a suppressor of DC maturation. The level of E2F1 expression was transiently downregulated in the course of LPS-induced maturation of both human monocyte-derived DCs and Downloaded from a mouse DC cell line, DC2.4. Knockdown of E2F1 by small interfering RNA in DC2.4 cells resulted in both phenotypic and functional maturation, even without LPS treatment. Conversely, ectopic overexpression of E2F1 suppressed LPS-induced maturation of DC2.4 cells. Furthermore, knockdown of E2F1 caused the activation of several major signaling pathways known to be activated in the course of DC maturation, including Erk1/2, NF-kB, and PI3K/Akt, suggesting that E2F1 may be involved in regulating multiple signaling pathways in DCs. Finally, the alteration of phenotypic maturation by E2F1 was confirmed with bone marrow-derived DCs from E2F1 knockout mice. Overall, our data demonstrate for the first time that E2F1 is a critical regulator of DC http://www.jimmunol.org/ maturation. The Journal of Immunology, 2010, 184: 6084–6091.

endritic cells (DCs) are the most potent professional APCs maturation involve the upregulation of surface costimulatory (e.g., that control immunity (1). Under steady state, immature CD80 and CD86) and MHC (class I and class II) molecules, the D DCs populate peripheral tissues where they continuously activation of lysosomal Ag-processing mechanisms, the production sample the environment by endocytosis. Upon encountering of numerous immunostimulatory cytokines, and the acquisition of pathogens and/or proinflammatory mediators, DCs undergo a com- the capacity to migrate to secondary lymphoid organs (1–3). Upon plex transformation process termed “maturation,” which greatly arriving at the secondary lymphoid organs, mature DCs present by guest on September 29, 2021 enhances their Ag-presenting capacity. The general features of DC antigenic peptides displayed on their surfaces in the context of MHC molecules to naive T cells for the initiation of Ag-specific T cell *Hefei National Laboratory for Physical Sciences at Microscale and School of Life immune responses, which is a unique capacity that only mature DCs Sciences, University of Science and Technology of China, Hefei, China; possess. Therefore, controlling the maturation of DCs is critical for † Laboratory of Molecular Immunoregulation, Cancer and Inflammation Program, the regulation of adaptive immune responses. Center for Cancer Research, National Cancer Institute, Frederick, MD 21701; and ‡Basic Science Program, Science Applications International Corporation-Frederick, DC maturation can be triggered by either exogenous agents, such National Cancer Institute at Frederick, Frederick, MD 21702 as pathogen-associated molecular patterns (e.g., microbial DNA, 1F.F. and Y.W. contributed equally to this work. RNA, LPS, etc.), or endogenous mediators, such as proinflammatory Received for publication August 7, 2009. Accepted for publication March 19, 2010. cytokines (e.g., TNF, IL-1, and IFN), CD40L, and alarmins. Most This work was supported by grants from the National Natural Science Foundation of pathogen-associated molecular patterns and alarmins induce DC China (30721002 and 30528020), National Basic Research Program of China (973 maturation by triggering certain pattern-recognition receptors, such Program) (2007CB914503), Ministry of Science and Technology of China (KSCX1- as TLRs (1, 4–7). Proinflammatory cytokines stimulate DC matu- YW-R-58 and KSCX2-YW-R-174), and China Ministry of Education (20060358019). Funding to pay the open access publication charges for this article was provided ration by signaling through their specific receptors, whereas CD40L by a grant from the National Basic Research Program of China (2007CB914503; to does so by interacting with CD40, a member of the TNFR super- W.X.). This research has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract N01- family on the DC surface (1, 2, 7–10). Despite utilization of a variety CO-12400. This research was supported in part by the Intramural Research Program of receptors, DC maturation in response to various ligands seems to of the National Institutes of Health, National Cancer Institute, Center for Cancer stem from the activation of a number of intracellular signaling Research. pathways including NF-kB, PI3K, and multiple MAPKs, which are The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, com- collectively responsible for inducing the phenotypic and functional mercial products, or organizations imply endorsement by the U.S. Government. characteristics typical of mature DCs (2, 3, 7–9, 11, 12). NF-kBisthe Address correspondence and reprint requests to Dr. Weihua Xiao, Institute of Immu- most frequently documented transcriptional factor, which plays nology, School of Life Sciences, University of Science and Technology of China, 443 critical roles in DC maturation (7, 10–13). Silencing RelB in DCs Huangshan Road, Hefei, China 230027, or Dr. De Yang, Basic Science Program, Science Applications International Corporation-Frederick, National Cancer Insti- using small interfering RNA (siRNA) has been found to not only tute at Frederick, Frederick, MD 21702. E-mail addresses: [email protected] or inhibit DC maturation but also downregulate DC-dependent de- [email protected] velopment of Ag-specific immune responses (13). Alymphoplasia Abbreviations used in this paper: Aly, alymphoplasia; ASK1, apoptosis signal-regu- (Aly) mouse has a loss-of-function point mutant in the NF-kB– lating kinase 1; DC, dendritic cell; KO, knockout; MDC, macrophage-derived chemokine; pcDNA, plasmid cDNA; siRNA, small interfering RNA; SLC, secondary inducing kinase (14), and consequently, all cells in Aly mice, in- lymphoid-tissue chemokine; [3H]TdR, [3H]thymidine deoxyribose; WT, wild-type. cluding DCs, are deficient in the activation of NF-kB via the www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902561 The Journal of Immunology 6085 noncanonical pathway (15). DCs of Aly mice show some deficiency presence of GM-CSF and IL-4 as described previously (29). Briefly, mon- 5 in maturation and are unable to present exogenous Ags to naive CD8 ocytes were incubated at 5 3 10 /ml in RPMI 1640 medium (RPMI 1640 T cells (15). Only sporadic reports are available with respect to the plus 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin, and 100 mg/ml streptomycin) containing 10% FBS (Haidmannweg 9; PAA Laboratories, participation of other transcriptional factors in the course of DC Pasching, Austria), 50 ng/ml recombinant human GM-CSF (PeproTech, maturation, such as CREB, activating transcription factor-2, and AP- Rocky Hill, NJ), and 50 ng/ml recombinant human IL-4 (PeproTech) at 1 (10, 11). 37˚C in a CO2 (5%) incubator for 5 d. The cultures were fed with the same Transcription factor E2F1 plays divergent roles in regulating gene cytokine-containing medium every 2–3 d. To induce DC maturation, DCs were cultured in the same cytokine mixtures in the presence of LPS (100 ng/ transcription, the cell cycle, and apoptosis (16). E2F1 induces cell ml) for an indicated period of time at 37˚C in a CO2 (5%) incubator. proliferation by advancing cells from the G0 phase to the S phase in the cell cycle (16–18). However, E2F1 can also induce apoptosis of DC2.4 culture and transfection different cell types through both p53-dependent and -independent DC2.4, an immature DC line in mice established previously (30), was ob- pathways (16, 19, 20). E2F12/2 mice exhibit increased lympho- tained from Dr. K. L. Rock (Dana-Farber Cancer Institute, Boston, MA). The proliferation and the development of various tumors including cells were cultured in DMEM supplemented with 10% FBS, 2 mM glutamine, 25 mM HEPES, 100 U/ml penicillin, 100 mg/ml streptomycin, and 50 lymphomas, suggesting that E2F1 has a proliferation-limiting role mM 2-ME at 37˚C with 5% CO2. For stimulation of maturation, the cells were in lymphocytes by acting as a tumor suppressor (21, 22). A more treated with LPS (1 mg/ml) for the indicated period of time before further recent study using E2F1 and E2F2 double-knockout (KO) mice not analysis. Transfection of DC2.4 cells was performed using Lipofectamine only demonstrated the profound effects of E2F1 and E2F2 on the 2000 (Invitrogen, Carlsbad, CA), according to the manufacturer’s in- proliferation and differentiation of lymphocytes and hematopoietic struction, and the stable-transfected cell lines were obtained by culturing transfected cells in the presence of 700 mg/ml G418 (Invitrogen) for 3–4 wk. progenitor cells but also revealed an increased sensitivity of T cells Downloaded from to antigenic stimulation as well as signs of autoimmunity in these Plasmids mice (23). Because Ag-specific lymphocyte proliferation in auto- The plasmids pU6-Scramble and pU6-siE2F1, which contain respectively immunity is likely initiated by mature, Ag-presenting DCs, the either a scrambled or a siRNA sequence against the E2F1 coding region, autoimmune activation of lymphocytes in E2F1 and E2F2 double- were described previously (31). The plasmid cDNA (pcDNA)3.1-E2F1 KO mice might be a result of the effects of E2F1 and E2F2 on DC expression plasmid was provided by Dr. M. Wu (University of Science and Technology of China, Hefei, China), the construction of which was de- development, differentiation, and/or maturation. Interestingly, the scribed previously (32). http://www.jimmunol.org/ activation of macrophages induced by LPS, a TLR4 agonist often Immunoblot analysis used for the stimulation of DC maturation, is accompanied by the recruitment of E2F1 to the promoters of many NF-kB–responsive For preparing the whole-cell extract, cells were harvested and lysed in genes (24). TNF, also capable of inducing DC maturation, has been radioimmunoprecipitation assay buffer (50 mM Tris-HCl [pH 7.4], 1% shown to inhibit endothelial cell proliferation by downregulating Nonidet P-40, 0.25% sodium deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 1 mg/ml pepstatin, 1 mM E2F1 in a manner dependent on JNK and Rb phosphorylation (25). Na3VO4, and 1 mM NaF). Protein concentration of the lysate was quan- In addition, E2F1, through transcriptional induction of Gab2 and tified by the use of a Bicinchoninic Acid Protein Assay Kit (Pierce, apoptosis signal-regulating kinase 1 (ASK1), respectively, regulates Rockford, IL). Cell lysates were loaded onto an SDS-PAGE gel and sep- the activation of PI3K/Akt and Erk1/2 signaling pathways (26, 27), arated by electrophoresis. After transfer of the proteins onto a piece of by guest on September 29, 2021 nitrocellulose membrane, the target molecules were detected by Western which are known to be activated in the course of DC maturation (2, blotting using corresponding primary Abs, HRP-conjugated secondary 9, 11). Nevertheless, the precise role of E2F1 in the course of DC Abs, and an ECL Detection kit (Pierce). maturation remains to be established. RT-PCR and real-time PCR analysis In the current study, we observed that the expression level of E2F1 was transiently downregulated during LPS-induced maturation of Total RNA was isolated using TRIzol reagent, according to the manu- human monocyte-derived DCs. Using DC2.4 (an immature DC cell facturer’s instruction. One microgram of total RNA was converted into cDNA with random primer and Superscript III reverse transcriptase (In- line in mice) as a model, we showed that depletion of E2F1 by siRNA vitrogen). PCR was performed with gene-specific primer sets: 59-GCCAC- resulted in DC maturation, even in the absence of LPS induction. In TGACTCTGCCACCATAG-39 and 59-CTGCCCATCCGGGACAAC-39 for contrast,ectopicoverexpressionofE2F1suppressedLPS-inducedDC human E2F1; and 59-TAGCCCTGGGAAGACCTCAT-39 and 59-CCCCA- maturation. Finally, bone marrow-derived DCs from E2F1 KO mice AAGTCACAGTCAAAGAG-39 for mouse E2F1. The RT-PCR products displayed a strongly enhanced maturation phenotype compared with were resolved on a 1.5% agarose gel and visualized by ethidium bromide staining. Quantitative PCR was performed with Sybr green incorporation on that of the control mice. Thus, our data demonstrate a potent sup- an ABI PRISM 7000, and the data were presented as mRNA accumulation pressive role for E2F1 in DC maturation, which is uniquely different index (2DDCt). from the known functions of E2F1 in the cell cycle and apoptosis. Flow cytometry Materials and Methods The expression levels of surface marker molecules were measured by Reagent and Abs immunostaining and flow cytometry using a FACSCalibur instrument (BD Biosciences, San Jose, CA). FACS data were analyzed using Win MDI 2.9 All chemicals and affinity-purified, HRP-conjugated anti-rabbit IgG, anti- software. For immunostaining, 1 3 106 cells were stained with FITC- or mouse IgG, and anti-goat IgG were purchased from Sigma-Aldrich (St. PE-conjugated Abs or isotype controls. The following PE-conjugated anti- Louis, MO). Abs against NF-kB p65 and NF-kB p50 were from Upstate mouse mAbs were used: anti-CD11c and anti-CD83 (eBioscience, San Biotechnology (Lake Placid, NY); Abs against E2F1, early growth response Diego, CA). The following FITC-conjugated anti-mouse mAbs were used: 1, transcription factor Dp-1, I-kBa, Akt, histone H1, and actin were pur- anti–HLA-DR (BD Biosciences) and anti-CD86 (eBioscience). All flow chased from Santa Cruz Biotechnology (Santa Cruz, CA). Abs against cytometric analyses were performed using appropriate isotype controls, 473 32/36 BCL2, BCL-XL, phospho-Akt (p-Ser ), and phospho–I-kBa (p-Ser ) and the data were collected for 10,000 cells/sample. The percentages of were from Cell Signaling Technology (Danvers, MA). positive cells were analyzed using a two-way factorial ANOVA in which LPS treatment and cell types (E2F1 siRNA and control) were the factors. Monocyte isolation and DC culture Migration assay Human monocytes were isolated from the peripheral blood of healthy adult volunteers using consecutive Ficoll-Hypaque and Percoll density gradient For in vitro studies, DC2.4 (105) in serum-free medium was placed in centrifugation as reported previously (28). The purity of monocytes was a Transwell migration chamber (Costar 3421; Corning Glass, Corning, ∼94% (6 2%) based on flow cytometric analysis of CD14 positivity. Human NY) and allowed to migrate through a polycarbonate membrane (pore size, monocyte-derived DCs were generated by culturing monocytes in the 5 mm) at 37˚C. Secondary lymphoid-tissue chemokine (SLC; 100 ng/ml) 6086 E2F1 SUPPRESSION OF DC MATURATION

(PeproTech) was placed in the lower chamber to induce cell chemotaxis. After 6 h, the wells were removed, and the cells on the top of the membrane were wiped off with a cotton swab. Cells on the bottom of the membrane were fixed with 3.7% formaldehyde for 15 min, stabbed with 0.5% Triton X-100 for 3 min, and stained with 1% Hochest at 37˚C for 6 min. The cells were counted using a light microscope. Images of cell migration under various conditions were photographed. The numeric data represented the means of three independent tests (each in triplicates) and SE (2pSE). For in vivo studies, DC2.4/Scramble and DC2.4/E2F1-KD cells were labeled with CFSE (5 mM, 37˚C, 20 min), washed, and re- suspended in PBS. One million (20 ml) CSFE-labeled DC2.4/Scramble and DC2.4/E2F1-KD cells were injected into the left and right hind footpads of C57BL/6 mice, respectively. After 36 h, popliteal lymph nodes were removed to make single-cell suspensions for the analysis of CSFE-positive cells by flow cytometry. Measurement of cytokine and chemokines Supernatants of cultured DCs were harvested and kept at 280˚C. The concentrations of selected cytokines and chemokines were measured using the multiplex protein array (Thermo Scientific, Rockford, IL) as described previously (6).

T cell proliferation Downloaded from Splenocytes were prepared from the spleens of 8-wk-old BALB/C mice. FIGURE 1. Transient downregulation of E2F1 in the course of LPS- Stimulator cells were grown on a 12-well plate in the presence or absence of induced maturation of human monocyte-derived DCs. Human monocyte- LPS (1 mg/ml) for 48 h. The cells were harvested, washed twice, sus- derived DCs were incubated in the absence or presence of LPS (100 ng/ml) 6 pended at 10 /ml, and irradiated at 8000 rad. The stimulator cells were for the period of time as indicated before harvest. The expression of surface ∼ diluted with the prepared splenocytes in a ratio of 1:5 to 1:3125 and marker molecules by DCs after 24 h of incubation in the absence (LPS[2]) 3 m cocultured for 4 d. [ H]Thymidine (1 Ci) was then added into each or presence (LPS[+]) of LPS was measured by flow cytometry (A). The culture well, and the culture wells were allowed to culture overnight. At http://www.jimmunol.org/ expression levels of E2F1 were assessed by both RT-PCR (B) or quantitative the end, the cells were harvested, and a liquid scintillation counter was used to measure the incorporation of [3H]thymidine. The data represent real-time PCR (C) with the use of total RNA isolated from DCs treated with three tests with triplicate samples in each test. LPS for various time periods. Similar levels of b-actin among all lanes indicate that a similar amount of mRNA template was used for the RT-PCR Generation, treatment, and analysis of bone marrow-derived amplification of E2F1 (B). Shown are from one experiment, which is rep- DCs in mice resentative of five performed, using monocytes from different donors. Bone marrow cells were flushed out from the tibias and femurs of 6- to ∼8- wk-old female E2F1 wild-type (WT) or KO mice (purchased from The histograms of flow cytometric analysis, indicating that LPS treat- Jackson Laboratory, Bar Harbor, ME). Bone marrow progenitors were ment was capable of inducing the maturation of DC2.4 cells. Sub- isolated and cultured in RPMI 1640 medium containing 20 ng/ml mouse by guest on September 29, 2021 GM-CSF (PeproTech) at 106/ml for 6 to ∼8 d to generate DCs as described sequently, the kinetics of E2F1 expression in DC2.4 cells in response previously (6). DCs obtained on day 8 were analyzed for the expression of to LPS-induced maturation was examined by RT-PCR (Fig. 2B), costimulatory and MHC class II molecules by flow cytometry. To compare real-time PCR (Fig. 2C), and Western blot analysis (Fig. 2D). Upon the maturational responses of WT and E2F1-KO DCs, DCs obtained on LPS treatment, E2F1 expression at the mRNA level in DC2.4 cells day 6 were cultured in the presence of LPS (200 ng/ml) for 22 h and exhibited a kinetic change similar to that of LPS-induced maturation subsequently analyzed by flow cytometry after immunostaining. of human monocyte-derived DCs: a transient downregulation be- Results ginning at 3 h, reaching its lowest level at 6 to ∼12 h and then the start E2F1 expression is transiently suppressed during LPS-induced of recovery at 18 h after LPS addition (Fig. 2B,2C). Western blot DC maturation analysis confirmed that upon LPS-induced maturation, E2F1 expression at the protein level in DC2.4 cells showed a similar transient As the first step in examining the role of E2F1 in DC maturation, we downregulation, albeit with a lag of 2 to ∼3 h (Fig. 2D). Therefore, determined whether the expression of E2F1 was altered in the course the expression of E2F1 was transiently suppressed during LPS- of maturation of human monocyte-derived DCs upon treatment with induced DC maturation in both human and mouse DCs, suggesting LPS. DCs treated with 100 ng/ml LPS for 24 h showed a much higher a potentially important role of E2F1 in DC maturation. expression of surface CD86, MHC class II, and CD83 (a critical marker of DC maturation) than DCs cultured in the absence of LPS, Knockdown of E2F1 enhances phenotypic maturation of DCs verifying that the concentration of LPS used was adequate in To further investigate the role of E2F1 in DC maturation, we inducing DC maturation (Fig. 1A). When monitored by RT-PCR, generated a DC2.4 cell line with its endogenous E2F1 knocked DCs in the course of LPS-induced maturation exhibited a time- down (designated hereafter as DC2.4/E2F1-KD) and compared its dependent change in E2F1 mRNA expression (Fig. 1B). E2F1 ex- phenotype with control cells (designated as DC2.4/Scramble). In pression began to decrease as early as 3 h, gradually reaching its comparison with DC2.4/Scramble cells, DC2.4/E2F1-KD cells lowest level at 12 h, and then started to recover at 18 to ∼24 h after exhibited a much lower E2F1 expression at both mRNA (Fig. 3A) LPS addition (Fig. 1B). The transient downregulation of E2F1 and protein levels (Fig. 3B), indicating that the knockdown was mRNA in the course of LPS-induced maturation of monocyte- effective. Interestingly, DC2.4/E2F1-KD cells showed remarkably derived DCs was also confirmed by quantitative PCR (Fig. 1C). higher expressions of surface MHC class II, CD86, CD83, and Tofacilitate investigationoftheroleofE2F1inDCmaturation,we CD11c than DC2.4/Scramble cells, suggesting that reducing the used DC2.4, an immature DC cell line established previously from level of E2F1 might promote maturation of DCs (Fig. 3C). The the culture of C57BL/6 bone marrow progenitors, as a model system upregulation of various surface markers on DC2.4/E2F1-KD is (30). As shown in Fig. 2A, surface expression of mouse MHC class unlikely to be the result of the transfection per se, because DC2.4/ II, CD86, CD83, and CD11c after treatment with 1 mg/ml LPS for Scramble cells had similar levels of surface costimulatory and 24 h was upregulated in DC2.4 cells as demonstrated by overlaying MHC class II expression as parental DC2.4 cells (data not shown). The Journal of Immunology 6087

FIGURE 2. Kinetic level of E2F1 during LPS-induced maturation of DC2.4 cells. DC2.4 cells were incubated without or with LPS (1 mg/ml) for the time specified in the induction of their maturation. The expression of certain surface marker molecules by DC2.4 cells after 24 h of incubation without [LPS (–)] or with [LPS (+)] LPS was determined by flow cytometry (A). E2F1 mRNA expression in the course of LPS-induced maturation of DC2.4 cells was measured by RT-PCR (B) or quantitative real-time PCR (C) with the use of total RNA isolated from DC2.4 cells treated with LPS for various time periods. Similar levels of b-actin Downloaded from among all lanes indicate that a similar amount of mRNA template was used for the RT-PCR amplification of E2F1 (B). The protein level of E2F1 in treated DC2.4 cells was detected by Western blot analysis using anti-E2F1 Ab as the probe (D). Similar levels of GAPDH proteins among all the lanes indicate that an almost identical amount of total proteins was loaded into each lane (D). Results shown are from one experiment, which is representative of four performed.

DC maturation is characterized by three phenotypic hallmarks: were found to produce higher levels of IL-12, TNF-a, Rantes, and 1) upregulation of surface costimulatory (e.g., CD83 and CD86) macrophage-derived chemokine (MDC) than the scramble control and MHC molecules; 2) increase in their capacity to produce cells in response to LPS stimulation (Fig. 3D). The kinetics of IL- http://www.jimmunol.org/ proinflammatory mediators (e.g., cytokines and chemokines); and 10 and IL-6 production were either similar or slightly delayed for 3) acquisition of the ability to respond to lymphoid tissue homing E2F1 knockdown cells (Fig. 3D). Of note, the basal level of IL-12 chemokine, such as ELC/CCL19 or SLC/CCL21 (1–3). To de- was significantly elevated in E2F1 knockdown DC2.4 cells in termine whether knockdown of E2F1 results in full maturation of comparison with the control cells, but this was not the case with DC2.4 cells or just upregulation of cell surface costimulatory and other tested cytokines. These data suggest that knockdown of MHC molecules, we investigated the capacity of DC2.4/E2F1-KD E2F1 enhances the capacity of DC2.4 cells to produce proin- cells to produce proinflammatory mediators and to migrate in flammatory cytokines (e.g., IL-12 and TNF-a) and chemokines

response to SLC/CCL21. After DC2.4/Scramble and DC2.4/ (e.g., Rantes and MDC) upon LPS-induced DC maturation. by guest on September 29, 2021 E2F1-KD cells were induced to mature with LPS and their pro- To examine the effect of E2F1 knockdown on DC migratory/ duction of selected cytokines and chemokines in the culture su- homing capacity, DC2.4/E2F1-KD and DC2.4/Scramble cells were pernatants was measured by protein array, DC2.4/E2F1-KD cells investigated both in vitro and in vivo for their migration in response

FIGURE 3. E2F1 knockdown in DC2.4 cells enhanced the expression of surface marker molecules and production of cytokines. DC2.4 cells were transfected with a vector expressing either siRNA specific for E2F1 or a scramble sequence (as control) and selected under G418 to establish two stable cell lines, DC2.4/E2F1-KD and DC2.4/Scramble. The E2F1 expression by these two cell lines was measured by RT-PCR at the mRNA level (A) and Western blot analysis at the protein level (B). The surface expression of selected marker molecules was determined by flow cytometry (C). The production of selected proinflammatory cytokines and chemokines in the supernatants was measured by multiplex protein array after stimulating the cells with LPS at 100 ng/ml for the time period specified (D). 6088 E2F1 SUPPRESSION OF DC MATURATION

FIGURE 4. Effect of E2F1 knockdown on the migratory capacity of DC2.4 cells. The in vitro migration of DC2.4/Scramble and DC2.4/E2F1- KD cells in response to SLC (100 ng/ ml) was determined by chemotaxis assay. Shown are the representative images (original magniﬁcation 3200) of cell migration under different conditions from one representative experiment (A) and the average (mean 6 2*SE) number of cell migrations from three separate experiments (B). To determine in vivo homing, CSFE-labeled DC2.4/Scramble and DC2.4/ E2F1-KD cells were injected into the left and right hind footpads of C57BL/ 6 mice, respectively. After 36 h, popliteal lymph nodes were removed to Downloaded from analyze the level of CSFE-positive cells by ﬂow cytometry (C).

to CCL21/SLC and for their homing ability to draining lymph vector (pcDNA) alone or with pcDNA-E2F1, followed by selection http://www.jimmunol.org/ nodes, respectively. A comparison of the migration of DC2.4/ with G418 to establish DC2.4/pcDNA and DC2.4/pcDNA-E2F1 cell E2F1-KD and DC2.4/Scramble cells in response to SLC/CCL21 lines. Overexpressions of E2F1 by DC2.4/pcDNA-E2F1 cells were revealed that SLC, at the concentration used, induced the migration confirmed by RT-PCR (Fig. 6A) and Western blot analysis (Fig. 6B). of DC2.4/E2F1-KD cells but not DC2.4/Scramble cells (Fig. 4A, When analyzed for the expression of surface marker molecules by 4B). In addition, analysis of accumulated CSFE-labeled DC2.4/ flow cytometry, DC2.4/pcDNA-E2F1 cells expressed much less E2F1-KD and DC2.4/Scramble cells injected into the left and MHC class II, CD86, and CD83 than DC2.4/pcDNA cells (Fig. 6C), right hind footpads of mice, respectively, in the popliteal lymph indicating that excessive E2F1 inhibited the maturation of DC2.4 nodes revealed that many DC2.4/E2F1-KD, but not DC2.4/ cells. In addition, DC2.4/pcDNA and DC2.4/pcDNA-E2F1 cells, Scramble, cells reached draining lymph nodes 36 h after injection either untreated or treated with LPS to stimulate maturation, were by guest on September 29, 2021 (Fig. 4C). These results indicate that E2F1 knockdown led to tested in allogeneic MLR to determine the effect of overexpression DC2.4’s acquisition of the capacity to migrate toward SLC in vitro of E2F1 on the functional maturation of DCs (Fig. 6D). DC2.4/ and to home in on draining lymph nodes in vivo. pcDNA cells matured by LPS treatment stimulated robust proliferation of allogeneic splenocytes at a DC:splenocyte ratio higher Knockdown of E2F1 enhances functional maturation of DCs than 1:125, suggesting that they could be activated into functionally Maturation of DCs is accompanied by the acquisition of enhanced mature DCs upon LPS treatment (Fig. 6D). In contrast, DC2.4/ capacity to present Ag(s) and migration (1, 2). To determine the effect of E2F1 knockdown in DC2.4 cells on their Ag-presenting capacity, we compared the capacity of DC2.4/E2F1-KD cells with DC2.4/Scramble cells to stimulate the proliferation of T cells in an allogeneic MLR assay. Splenocytes from BALB/c mice were used as responder T cells because of the C57BL/6 origin of DC2.4 cells. DC2.4/Scramble cells had a very low stimulatory capacity (Fig. 5, ♦). However, DC2.4/Scramble treated with LPS, as expected, de- veloped the capacity to stimulate the proliferation of allogeneic T cells (Fig. 5, N). DC2.4/E2F1-KD cells (Fig. 5, d) exhibited a remarkable enhanced capacity to stimulate the proliferation of allogeneic T cells as compared with DC2.4/Scramble cells (Fig. 5, ♦), indicating that knockdown of E2F1 greatly enhanced the Ag- presenting capacity of DC2.4. Of note, LPS treatment of DC2.4/ FIGURE 5. E2F1 knockdown resulted in the functional maturation of E2F1-KD cells (Fig. 5, 4) did not enhance their capacity to stim- DC2.4 cells. DC2.4/Scramble and DC2.4/E2F1-KD cells were incubated in ulate the proliferation of allogeneic T cells (Fig. 5, compare 4 with the absence or presence of LPS (1 mg/ml) for 48 h before they were har- d), suggesting that knockdown of E2F1 caused full maturation of vested and gamma irradiated. Subsequently, various numbers of BALB/c splenocytes in the indicated ratio with irradiated DC2.4 cells (5 3 105) were DC2.4 at the functional level. Therefore, knockdown of E2F1 re- mixed and added into a 96-well, flat-bottomed, tissue culture plate (in sulted in both phenotypic and functional maturation of DC2.4 cells. triplicate). The plate was incubated at 37˚C in humidified air containing 5% 3 m Overexpression of E2F1 suppresses DC maturation CO2 for 96 h, with the addition of [ H]TdR (0.5 Ci/well) for the last 18 h. The cells were harvested and the incorporation of [3H]TdR was measured by To further demonstrate the suppressive effect of E2F1 on DC beta scintillation counting. Shown is the average cpm (means 6 SD) of maturation, we also determined the effect of E2F1 overexpression on triplicate wells of one experiment representative of three. [3H]TdR, [3H] DC phenotype and function. DC2.4 cells were transfected with thymidine deoxyribose. The Journal of Immunology 6089

FIGURE 6. Effect of E2F1 overexpression on the phenotype and function of DC2.4 cells. DC2.4 cells were transfected with a pcDNA vector or pcDNA- E2F1 expression plasmid and selected by G418 to establish DC2.4/pcDNA and DC2.4/pcDNA-E2F1 cell lines. The overexpression of E2F1 in DC2.4/ Downloaded from pcDNA-E2F1 cells were conﬁrmed at the mRNA level by RT-PCR (A) and at the protein level by Western blot analysis (B). The expression of certain costimulatory and MHC class II molecules on the surface of these cells was measured by ﬂow cytometry after staining with appropriate Abs (C). To determine the Ag-presenting capacity of DC2.4/pcDNA and DC2.4/pcDNA-E2F1 cells, they were either treated with or without LPS and subsequently assayed by an allogeneic MLR assay as detailed in the legend of Fig. 4. Shown is the average cpm (mean 6 SD) of triplicate wells of one experiment representative of two (D). http://www.jimmunol.org/ pcDNA-E2F1 cells, even after LPS treatment, did not stimulate the expression (Fig. 7B) of E2F1 together with their corresponding proliferation of allogeneic mouse lymphocytes at any DC:spleno- controls were subjected to Western blot analysis. As shown in Fig. 6, cyte ratios tested, indicating that overexpression of E2F1 sup- the activation of several intracellular signal transducers (as indicated pressed LPS-induced functional maturation of DC2.4 cells (Fig. by the level of phosphorylated proteins) including Erk1/2, Akt, and 6D). Thus, data from both knockdown and overexpression experi- NF-kB p65 were markedly enhanced by E2F1 knockdown as ments supported the notion that E2F1 appears to be a critical sup- compared with control cells (Fig. 7A). Conversely, their levels of pressor of DC maturation. activation in DC2.4 cells were reduced upon E2F1 overexpression (Fig. 7B). In contrast, the level of phospho-p38 was decreased in

Alteration of E2F1 in DC2.4 cells inﬂuences multiple signaling by guest on September 29, 2021 E2F1 knockdown cells but increased in E2F1 overexpression cells, pathways in comparison with corresponding levels in the control cells. E2F1 Maturation of DC involves a complicated cellular transition process knockdown or overexpression did not appear to affect the expression that is associated with the activation of multiple signaling pathways. of unphosphorylated ERKs, p38, Akt, and NF-kB p65, as evidenced To gain some insight into how E2F1 suppresses DC maturation, by similar bands between E2F1 knockdown or overexpressed several major signaling pathways that are considered critical for DC maturation were examined. The cell lysate from two sets of stably transfected DC2.4 cells with either knockdown (Fig. 7A) or over-

FIGURE 8. Comparative analysis of the maturational status of DCs generated from E2F1 WT and KO mice bone marrow progenitors. DCs were differentiated from bone marrow mononuclear cells by culturing in FIGURE 7. Effect of E2F1 knockdown or overexpression on the acti- the presence of 20 ng/ml mouse GM-CSF for 6 to ∼8d.A, BM-derived vation of multiple signal transducers in DC2.4 cells. Western blot analysis DCs on day 8 (from both WT and KO) were immunostained with FITC- or of the total and phosphorylated forms of selected intracellular signal PE-conjugated Abs against mouse CD83, CD80, CD86, or MHC class II transducers (including ERKs p38, Akt, and NF-kBp65) were performed (closed area) or isotype-matched control Abs (open area) and analyzed by with cell lysate from two sets of DC2.4 cells with either E2F1-knockdown ﬂow cytometry. B, Bone marrow-derived DCs on day 6 were treated with (A) or E2F1 overexpression (B) and their corresponding control cells that 200 ng/ml LPS for 22 h before they were stained and analyzed as in A. The were established and used in Fig. 6. Lane 1, DC2.4/Scramble; lane 2, numbers inside the histograms are the geometric mean of relative ﬂuo- DC2.4/E2F1-KD; lane 3, DC2.4/pcDNA; and lane 4, DC2.4/pcDNA- rescence of corresponding surface molecules. Shown are the overlay his- E2F1. GAPDH was used as a loading control. tograms of one experiment representative of two. 6090 E2F1 SUPPRESSION OF DC MATURATION

DC2.4’s with their corresponding control cells (Fig. 7). Thus, E2F1 activation in DC2.4 cells was reduced by E2F1 knockdown and regulates the activation of many signal transduction pathways that enhanced by E2F1 overexpression (Fig. 7). Although it is unclear are associated with DC maturation. why this seemingly paradoxical phenomenon occurred in DC2.4 cells, it might be due to the direct promoting effect of E2F1 on the E2F1 KO DCs show a more mature phenotype activation of p38 MAPK. In this regard, several previous studies Todetermine the physiologic relevance of our findings,wegenerated using DNA screening microassays have shown that E2F1 upregu- DCs from the bone marrow of E2F1 KO and WT control mice and lates the level of ASK1, a member of the MAPK kinase kinase measured the expression of a number of surface molecules. E2F1 KO family capable of activating the p38 MAPK pathway in diverse cell DCs cultured in vitro for 8 d exhibited higher expression of CD86, types (34–36). Recently, E2F1 has been shown to promote the ac- CD80, MHC class II, and CD83 than E2F1 WT DCs generated under tivation of p38 MAPK through induction of ASK1 in both osteo- identical condition (Fig. 8A). Moreover, when day 6 DCs were sarcoma cells and human lung fibroblasts (27). It remains to be treated with LPS for 22 h, E2F1 KO DCs upregulated higher levels determined whether E2F1 indeed regulates the activity of ASK1 in of all surface molecules measured than identically treated E2F1 DC2.4 cells. WT DCs (Fig. 8B). These data are consistent with the results of The E2F1 transcription factor plays diverse roles in cells ranging E2F1 knockdown or overexpression experiments and collectively from promoting cell proliferation in certain cell types to promoting demonstrate the suppressive effect of E2F1 on DC maturation. cell death by apoptosis in other cell types (16–20, 22, 31). The identification of E2F1 as a suppressor of DC maturation in the Discussion current study adds another role to the panoply of functions ascribed

The term “DC maturation,” originally coined to define the acqui- to E2F1. The participation of E2F1 in DC maturation is suggested Downloaded from sition of Ag-presenting activity by murine epidermal Langerhans by a very recent paper reporting that E2F1 and several other tran- cells after isolation from skin (33), is used to describe the overall scription factors including NF-kB, AP-1, AP-2, SP1, and early phenotypic and functional transformations of DCs triggered by di- growth response 1 are involved in the activation of monocytes and verse exogenous and endogenous mediators, such as TLR ligands DCs induced by alarmin LL-37 (37). The identification of E2F1 as (e.g., LPS), proinflammatory cytokines, alarmins, and CD40L a suppressor of DC maturation suggests that E2F1 may act as an

(1–5). Despite advances in studies on DC maturation, the identity of anti-inflammatory or immunosuppressive transcription factor. In http://www.jimmunol.org/ the transcription factors that play critical roles in the course of DC this regard, E2F1 has previously been shown to suppress TNF-a– maturation is still not fully understood. Previous studies have shown induced activation of human aortic endothelial cells by inhibiting that several transcription factors including NF-kB, AP-1, activating NF-kB activation and translocation (38). Furthermore, mice lack- transcription factor-2, and CREB, are involved in the course of DC ing E2F1 are reported to be more susceptible to the development of maturation (7, 9, 10, 12, 13, 15). Our present study demonstrates that autoimmune syndromes (23, 39, 40). In these mice, there are ex- E2F1 is another transcription factor that controls DC maturation. cessive numbers of mature CD4 and CD8 T cells and reduced First, in the course of LPS-induced maturation of human monocyte- number of CD4+/CD25+ regulatory T cells, which is thought to be derived DCs, as well as mouse DC2.4 DCs, the level of E2F1 un- to the result of a defect in the apoptosis of T cells (23, 40). However, derwent a transient downregulation in maturing DCs both at the with the identification of E2F1 as a suppressor of DC maturation in by guest on September 29, 2021 mRNA and protein levels (Figs. 1, 2). In addition, knockdown of the current study, we can speculate that lack of E2F1 could augment E2F1 expression in DC2.4 cells modified their phenotypic and maturation of DCs carrying self-Ags and enhance the activation of functional features to those of mature DCs, including upregulation autoreactive lymphocytes, reducing the generation of inducible of surface costimulatory and MHC molecules, enhanced production regulatory T cells in E2F1 KO mice. of various proinflammatory cytokines (e.g., IL-12p70 and TNF-a) and chemokines (e.g., Rantes and MDC), acquisition of the capacity Acknowledgments to respond to lymphoid-homing chemokine SLC to allow them to We thank Dr. Joost J. Oppenheim (Laboratory of Molecular Immunoreg- home in on draining lymph nodes, and augmented Ag-presenting ulation, Cancer and Inflammation Program, National Cancer Institute at capacity (Figs. 3–5). Furthermore, overexpression of E2F1 in DC2.4 Frederick, MD) for helpful discussion and critical reading of the manu- cells decreased their surface expression of maturational markers and script. LPS-induced enhancement of Ag-presenting capacity (Fig. 6). Al- though unsuccessful attempts were made to do knockdown with Disclosures primary DCs due mainly to low transfection rates, nevertheless, The authors have no financial conflicts of interest. E2F1 KO DCs showed a more mature phenotype than E2F1 WT DCs, demonstrating the physiologic relevance of our findings (Fig. References 8). Therefore, E2F1 acts as a suppressor of DC maturation and, to 1. Banchereau, J., and R. M. Steinman. 1998. Dendritic cells and the control of our knowledge, is the first transcription factor to be identified as immunity. Nature 392: 245–252. a negative regulator of DC maturation. 2. Mellman, I. 2005. Antigen processing and presentation by dendritic cells: cell biological mechanisms. Adv. Exp. Med. Biol. 560: 63–67. The maturation of DCs in response to numerous stimuli is ac- 3. Randolph, G. J., V. Angeli, and M. A. Swartz. 2005. Dendritic-cell trafficking to companied by the activation of several intracellular signaling lymph nodes through lymphatic vessels. Nat. Rev. Immunol. 5: 617–628. transducers including NF-kB, PI3K, and members of the MAPK 4. Iwasaki, A., and R. Medzhitov. 2004. Toll-like receptor control of the adaptive immune responses. Nat. Immunol. 5: 987–995. superfamily (2, 6–9, 11, 12). In support of the role of E2F1 as 5. Oppenheim, J. J., and D. Yang. 2005. Alarmins: chemotactic activators of im- a suppressor of DC maturation, knockdown of E2F1 in DC2.4 cells mune responses. Curr. Opin. Immunol. 17: 359–365. 6. Yang, D., Q. Chen, S. B. Su, P. Zhang, K. Kurosaka, R. R. Caspi, resulted in the activation of ERKs Akt (indicative of PI3K activa- S. M. Michalek, H. F. Rosenberg, N. Zhang, and J. J. Oppenheim. 2008. tion) and NF-kB, whereas overexpression of E2F1 in DC2.4 cells Eosinophil-derived neurotoxin acts as an alarmin to activate the TLR2-MyD88 resulted in a reduction in their activation (Fig. 7). Because p38 signal pathway in dendritic cells and enhances Th2 immune responses. J. Exp. Med. 205: 79–90. MAPK is also known to be activated during DC maturation, it was 7. Lyakh, L. A., G. K. Koski, W. Telford, R. E. Gress, P. A. Cohen, and N. R. Rice. therefore expected that knockdown of E2F1 (by inducing DC mat- 2000. Bacterial lipopolysaccharide, TNF-a, and calcium ionophore under serum- + uration) would enhance, whereas E2F1 overexpression would de- free conditions promote rapid dendritic cell-like differentiation in CD14 monocytes through distinct pathways that activate NK-kB. J. Immunol. 165: crease, the activation of p38 MAPK in DC2.4 cells. Surprisingly, its 3647–3655. The Journal of Immunology 6091

8. Arrighi, J. F., M. Rebsamen, F. Rousset, V. Kindler, and C. Hauser. 2001. A (p65) binding identiﬁes E2F1 as a transcriptional activator recruited by NF-kB critical role for p38 mitogen-activated protein kinase in the maturation of human upon TLR4 activation. Mol. Cell 27: 622–635. blood-derived dendritic cells induced by lipopolysaccharide, TNF-a, and contact 25. Kishore, R., C. Luedemann, E. Bord, D. Goukassian, and D. W. Losordo. 2003. sensitizers. J. Immunol. 166: 3837–3845. Tumor necrosis factor-mediated E2F1 suppression in endothelial cells: differ- 9. Yu, Q., C. Kovacs, F. Y. Yue, and M. A. Ostrowski. 2004. The role of the p38 ential requirement of c-Jun N-terminal kinase and p38 mitogen-activated protein mitogen-activated protein kinase, extracellular signal-regulated kinase, and kinase signal transduction pathways. Circ. Res. 93: 932–940. phosphoinositide-3-OH kinase signal transduction pathways in CD40 ligand- 26. Chaussepied, M., and D. Ginsberg. 2004. Transcriptional regulation of AKT induced dendritic cell activation and expansion of virus-speciﬁc CD8+ T cell activation by E2F. Mol. Cell 16: 831–837. memory responses. J. Immunol. 172: 6047–6056. 27. Hershko, T., K. Korotayev, S. Polager, and D. Ginsberg. 2006. E2F1 modulates 10. Mann, J., F. Oakley, P. W. Johnson, and D. A. Mann. 2002. CD40 induces in- p38 MAPK phosphorylation via transcriptional regulation of ASK1 and Wip1. J. terleukin-6 gene transcription in dendritic cells: regulation by TRAF2, AP-1, Biol. Chem. 281: 31309–31316. NF-kB, and CBF1. J. Biol. Chem. 277: 17125–17138. 28. Yang, D., Q. Chen, A. P. Schmidt, G. M. Anderson, J. M. Wang, J. Wooters, 11. Ardeshna, K. M., A. R. Pizzey, S. Devereux, and A. Khwaja. 2000. The PI3 J. J. Oppenheim, and O. Chertov. 2000. LL-37, the neutrophil granule- and epi- kinase, p38 SAP kinase, and NF-kB signal transduction pathways are involved in thelial cell-derived cathelicidin, utilizes formyl peptide receptor-like 1 (FPRL1) the survival and maturation of lipopolysaccharide-stimulated human monocyte- as a receptor to chemoattract human peripheral blood neutrophils, monocytes, and derived dendritic cells. Blood 96: 1039–1046. T cells. J. Exp. Med. 192: 1069–1074. 12. Rescigno, M., M. Martino, C. L. Sutherland, M. R. Gold, and P. Ricciardi- 29. Yang, D., H. F. Rosenberg, Q. Chen, K. D. Dyer, K. Kurosaka, and J. J. Oppenheim. Castagnoli. 1998. Dendritic cell survival and maturation are regulated by dif- 2003. Eosinophil-derived neurotoxin (EDN), an antimicrobial protein with che- ferent signaling pathways. J. Exp. Med. 188: 2175–2180. motactic activities for dendritic cells. Blood 102: 3396–3403. 13. Li, M., X. Zhang, X. Zheng, D. Lian, Z. X. Zhang, W. Ge, J. Yang, C. Vladau, 30. Shen, Z., G. Reznikoff, G. Dranoff, and K. L. Rock. 1997. Cloned dendritic cells M. Suzuki, D. Chen, et al. 2007. Immune modulation and tolerance induction by can present exogenous antigens on both MHC class I and class II molecules. J. RelB-silenced dendritic cells through RNA interference. J. Immunol. 178: 5480– Immunol. 158: 2723–2730. 5487. 31. Zheng, C., Z. Ren, H. Wang, W. Zhang, D. V. Kalvakolanu, Z. Tian, and W. Xiao. 14. Shinkura, R., K. Kitada, F. Matsuda, K. Tashiro, K. Ikuta, M. Suzuki, K. Kogishi, 2009. E2F1 Induces tumor cell survival via nuclear factor-kB–dependent in- T. Serikawa, and T. Honjo. 1999. Alymphoplasia is caused by a point mutation in duction of EGR1 transcription in prostate cancer cells. Cancer Res. 69: 2324– the mouse gene encoding Nf-kb-inducing kinase. Nat. Genet. 22: 74–77. 2331. Downloaded from 15. Lind, E. F., C. L. Ahonen, A. Wasiuk, Y. Kosaka, B. Becher, K. A. Bennett, and 32. Xie, W., P. Jiang, L. Miao, Y. Zhao, Z. Zhimin, L. Qing, W. G. Zhu, and M. Wu. R. J. Noelle. 2008. Dendritic cells require the NF-kB2 pathway for cross- 2006. Novel link between E2F1 and Smac/DIABLO: proapoptotic Smac/DIA- presentation of soluble antigens. J. Immunol. 181: 354–363. BLO is transcriptionally upregulated by E2F1. Nucleic Acids Res. 34: 2046–2055. 16. DeGregori, J., and D. G. Johnson. 2006. Distinct and overlapping roles for E2F 33. Schuler, G., and R. M. Steinman. 1985. Murine epidermal Langerhans cells family members in transcription, proliferation and apoptosis. Curr. Mol. Med. 6: mature into potent immunostimulatory dendritic cells in vitro. J. Exp. Med. 161: 739–748. 526–546. 17. Johnson, D. G., J. K. Schwarz, W. D. Cress, and J. R. Nevins. 1993. Expression 34. Mu¨ller, H., A. P. Bracken, R. Vernell, M. C. Moroni, F. Christians, E. Grassilli,

of transcription factor E2F1 induces quiescent cells to enter S phase. Nature 365: E. Prosperini, E. Vigo, J. D. Oliner, and K. Helin. 2001. E2Fs regulate the ex- http://www.jimmunol.org/ 349–352. pression of genes involved in differentiation, development, proliferation, and 18. Wu, L., C. Timmers, B. Maiti, H. I. Saavedra, L. Sang, G. T. Chong, F. Nuckolls, apoptosis. Genes Dev. 15: 267–285. P. Giangrande, F. A. Wright, S. J. Field, et al. 2001. The E2F1-3 transcription 35. Stanelle, J., T. Stiewe, C. C. Theseling, M. Peter, and B. M. Pu¨tzer. 2002. Gene factors are essential for cellular proliferation. Nature 414: 457–462. expression changes in response to E2F1 activation. Nucleic Acids Res. 30: 1859– 19. Wu, X., and A. J. Levine. 1994. p53 and E2F-1 cooperate to mediate apoptosis. 1867. Proc. Natl. Acad. Sci. USA 91: 3602–3606. 36. Jamshidi-Parsian, A., Y. Dong, X. Zheng, H. S. Zhou, W. Zacharias, and 20. Nahle, Z., J. Polakoff, R. V. Davuluri, M. E. McCurrach, M. D. Jacobson, K. M. McMasters. 2005. Gene expression proﬁling of E2F-1–induced apoptosis. M. Narita, M. Q. Zhang, Y. Lazebnik, D. Bar-Sagi, and S. W. Lowe. 2002. Direct Gene 344: 67–77. coupling of the cell cycle and cell death machinery by E2F. Nat. Cell Biol. 4: 37. Mookherjee, N., P. Hamill, J. Gardy, D. Blimkie, R. Falsaﬁ, A. Chikatamarla, 859–864. D. J. Arenillas, S. Doria, T. R. Kollmann, and R. E. Hancock. 2009. Systems 21. Yamasaki, L., T. Jacks, R. Bronson, E. Goillot, E. Harlow, and N. J. Dyson. 1996. biology evaluation of immune responses induced by human host defence peptide

Tumor induction and tissue atrophy in mice lacking E2F-1. Cell 85: 537–548. LL-37 in mononuclear cells. Mol. Biosyst. 5: 483–496. by guest on September 29, 2021 22. Field, S. J., F. Y. Tsai, F. Kuo, A. M. Zubiaga, W. G. Kaelin, Jr., 38. Chen, M., C. Capps, J. T. Willerson, and P. Zoldhelyi. 2002. E2F-1 regulates D. M. Livingston, S. H. Orkin, and M. E. Greenberg. 1996. E2F-1 functions in nuclear factor-kB activity and cell adhesion: potential antiinflammatory activity mice to promote apoptosis and suppress proliferation. Cell 85: 549–561. of the transcription factor E2F-1. Circulation 106: 2707–2713. 23. Zhu, J. W., S. J. Field, L. Gore, M. Thompson, H. Yang, Y. Fujiwara, 39. Salam, M. A., K. Matin, N. Matsumoto, Y. Tsuha, N. Hanada, and H. Senpuku. R. D. Cardiff, M. Greenberg, S. H. Orkin, and J. DeGregori. 2001. E2F1 and 2004. E2f1 mutation induces early onset of diabetes and Sjo¨gren’s syndrome in E2F2 determine thresholds for antigen-induced T-cell proliferation and suppress nonobese diabetic mice. J. Immunol. 173: 4908–4918. tumorigenesis. Mol. Cell. Biol. 21: 8547–8564. 40. Marıń, M. J., I. Garcıá, M. Penã, A. Bolı´var, A. Zubiaga, and M. Loéz-Hoyos. 24. Lim, C. A., F. Yao, J. J. Wong, J. George, H. Xu, K. P. Chiu, W. K. Sung, 2005. E2F1–/– C57BL/6 mice overexpressing a human Bcl-2 transgene in B cells L. Lipovich, V. B. Vega, J. Chen, et al. 2007. Genome-wide mapping of RELA develop a mild autoimmune syndrome. Ann. N. Y. Acad. Sci. 1051: 156–165.